The present invention relates to the propagation of animal cells for the purpose of recovering cell-secreted products of interest therefrom, and more particularly to an apparatus and process for the large-scale propagation of animal cells for such purpose.
It has long been known that animal cells, particularly human cells, secrete in vivo a wide variety of protein products which, directly or indirectly, are responsible for effecting particular functions or controlling or regulating particular functions in the species, including products such as hormones, enzymes, antibodies, clotting agents, and the like. It also has long been known that such naturally-secreted products, if capable of being isolated and recovered from an animal, offer enormous potential in the diagnostic and therapeutic fields.
Unfortunately, the amounts of these naturally-occurring products which are secreted by the cells of animals are generally very small, and isolation of such products per se, let alone in any useful quantity, is extraordinarily difficult and expensive.
It is generally accepted that a preferred route to production of useful quantities of natural cell-secreted products is by means of in vitro culture of the very animal cells which produce such products in vivo. While many natural product-secreting animal cells are not capable of sustained growth and subdivision in culture, there does exist a number of such cells (e.g., melanomas) which do possess this capability and which thus can be grown up in culture to cell densities at which useful quantities of their secreted products can be recovered. It is now also possible to "immortalize" animal cells which secrete a product of interest, but which otherwise are incapable of sustained growth and subdivision in culture, by, e.g., fusing them to a partner (e.g., a myeloma cell) which does possess such capability and which confers that capability to the hybrid cell, thereby enabling the in vitro culture of the hybrid cell line to produce significant quantities of the secreted product. Even in the realm of recombinant DNA technology, where heterologous genes coding for a particular product of interest are used to transform host cells, in vitro animal cell culture is of ever increasing importance due to the desire to employ animal cells as host cells in an effort to achieve recombinantly-produced products which are more like their naturally-secreted counterparts in terms of glycosylation and other structural/functional characteristics, and which are not at risk of being produced in association with undesirable products as could be the case utilizing bacteria as host cells.
The mere fact that a product-secreting cell, either naturally or by manipulation, possesses the inherent ability to undergo generally continuous growth and subdivision is, of course, insufficient by itself to enable its culture in vitro so as to produce and recover secreted product. The in vitro environment must be such as to provide the cells with the nutrition, oxygenation and other like requirements which induce and permit the cells to reproduce and secrete product.
Most successful in vitro animal cell culture devices and processes are of fairly small scale and are capable of producing only limited quantities of cell-secreted products. In many of these systems, the successful provision of a suitable environment for cell growth and product secretion is in part a consequence of the small scale, i.e., because the dimensions of the system are not very large, and also since the cell densities are often not extremely high, it is possible to provide an environment which is generally homogeneous in terms, e.g., of the concentration of nutrients and/or gases, and to which a large number of the cells can be exposed. Examples of such small-scale animal cell culture techniques and devices include roller bottles, stirred flask reactors, small hollow-fiber bioreactors and the like. In devices of this type, whether used for growth of anchorage-dependent cells or cells which can be grown in suspension, the rotation, stirring or short flow paths, often coupled with reasonably low cell densities, permits the establishment of a reasonably homogeneous environment capable of providing the requisite nutrition to most of the cells.
As more cell-secreted products of interest have become identified as of late, and as more uses for such products in diagnosis and therapy have been postulated and/or demonstrated, the demand is ever-increasing for production of such products in significant quantities. One obvious means for meeting this demand via in vitro animal cell culture is to make use of the heretofore proven small-scale methodologies and devices and simply increase the number of such units such that, in toto, many more cells are cultured and more secreted product produced. Among the difficulties in proceeding in this manner is that each such unit must be separately seeded, separately fed and gassed, separately observed and maintained, and separately processed to collect therefrom culture fluid containing secreted cell product, all of which adds enormously to the labor and equipment required and, hence, to the production cost (e.g., per gram) of the product of interest.
At least in theory, a far more economical route to large-scale production of secreted cell products via animal cell culture is simply to increase (i.e., scale-up) the capacity of a proven small-scale culture device such that it can accommodate many more cells and generate correspondingly larger quantities of product. Scale-up of these small-scale culture units has generally been quite unsuccessful, however, i.e., their capacity cannot be increased cost effectively while still maintaining the process and product-producing efficiencies demonstrated in the small-scale units. In large part, this inability is due to the difficulty of achieving any reasonable degree of homogeneity in the in vitro environment per se, and the further inability to expose even a majority of cells in high density clusters to the requisite nutrition and gas components of the culture medium.
Certain cell culture designs have demonstrated at least some degree of scaleability, such as air-lift reactors, stirred tank reactors and fluidized bed reactors. For large-scale culturing of anchorage-dependent cells, the stirred tank reactor and fluidized bed reactor can be employed by propagating the cells on or within solid carriers which, in turn, are maintained in suspension in the in vitro environment. However, these carrier systems are not presently capable of supporting the growth of all anchorage-dependent cell lines of interest and, in scaled-up systems, often-times exhibit reduced levels of product secretion as compared to that found in small, laboratory-scale units.
For growth of anchorage-dependent cells, glass bead packed bed reactors have been known for some time, and our own studies have demonstrated that such reactors, in small scale (i.e., from about 1 to 4 liter bead volume), are highly effective for use in continuous culture both of anchorage-dependent cells, where the cells attach to and grow on the bead surfaces, and of cells which do not require attachment for growth, where the cells are confined within and grow in the void spaces of the packed bed. Additional advantages of this reactor design are that it can be inoculated at relatively low cell densities (e.g., as low as 1/100 of the final cell density), thereby permitting use of a small number of seed vessels and related manipulations, while attaining and supporting high cell densities over long periods of time.
Unfortunately, despite indication in the art that glass bead packed bed culture units can be scaled up to industrial levels (an indication based upon results of short term batch propagation of particular cells to produce a particular virus), our studies have demonstrated that such reactors pose quite significant scale-up problems when long-term continuous culture is desired. As the glass bead bed height is increased to provide either greater surface area for attachment and growth, or greater void space for accommodation and growth, of the large number of anchorage-dependent or non-anchorage-dependent cells required for industrial-scale processing, medium to be perfused through the bed for nourishing the cells becomes increasingly less capable of achieving that result by reason of increasing depletion of the nutrients or gases therein over the length of the bed as they are consumed by the cells. As a consequence, significant cell density gradients exist throughout the length of the bed. In addition, uneven distribution of cells or cellular material throughout the bed brings about channelling and by-passing of medium through the bed (i.e., the seeking out of preferential flow paths offering least resistance), leading to non-uniform growth conditions.
In short, glass bead packed bed reactor units can easily be scaled-up in size per se (i.e., made larger), but only at a significant loss of process and product-production efficiency and at a significant loss in the ability to easily translate to large scale, if at all, the nutrition and other growth and product-secretion parameters determined to be optimum for a particular cell line in small-scale laboratory tests. Without this latter ability, each scale-up ends up requiring a comprehensive development program to reestablish operable and/or optimum growth conditions, nutrient requirements and the like, adding enormously to the costs of production when just the opposite is desired.